7. Project Summary/Abstract Sex differences in the liver transcriptome are widespread in both mice and humans and are largely regulated by growth hormone (GH). The long-term goal of this project is to elucidate these sex differences to better understand the mechanistic underpinnings of the many clinically relevant sex differences impacted by GH; these include sex- differences in hepatic drug and steroid metabolism and lipid metabolic profiles, and in the incidence and severity of liver pathologies, such as non-alcoholic fatty liver disease (NAFLD) and liver fibrosis associated with development of hepatocellular carcinoma. Our recent studies in the mouse model revealed that GH acts through its sex-specific temporal patterns of pituitary secretion ? pulsatile in males and persistent in females ? and via GH- stimulated activation of liver STAT5, to establish a sex-differential epigenomic environment that enables the sex- specific actions of GH in the liver. We identified several thousand genomic regions marked as putative enhancers that have sex-biased binding sites for STAT5 and other essential GH-regulated liver transcription factors; and we showed that sex-specific deposition by Ezh1/Ezh2 of histone-H3 lysine 27 trimethyl marks (H3K27me3) is required specifically for the repression of many female-biased genes in male liver. Further, more than 200 sex-specific, GH- regulated and nuclear-enriched long, non-coding RNAs (lncRNAs) were discovered, and strong candidates for regulation of the sex-differential deposition of H3K27me3 and other chromatin marks at sex-specific genes and their enhancers were identified by analysis of a large panel of Diversity Outbred mouse livers. This project builds on these advances to elucidate fundamental mechanisms that underlie the transcriptional and epigenetic regulation by GH of sex-biased gene expression essential for normal liver function. The work proposed has two major aims: 1) to discover critical features that underpin sex-biased gene transcription associated with sex-biased liver disease by identifying functionally active sex-biased enhancers, which harbor the majority of genetic risk factors for fatty liver disease, and to elucidate their organization within chromatin loop domains and subdomains, and their interactions with sex-biased gene promoters; and 2) to discover the role of sex-specific, GH-regulated lncRNAs in establishing and maintaining the sex-differentiated chromatin states at sex-biased enhancers and genes to support sex differences in liver gene transcription, and then elucidate their contributions to the protective effects of GH- activated STAT5 against hepatic stresses that induce non-alcoholic fatty liver disease and other liver pathologies. Together, this work will identify key mechanistic features that enable GH, and its sex-dependent plasma patterns, to regulate the sex-biased expression of hundreds of genes that control liver metabolic processes with a major impact on human health and liver disease, and may link molecular features to pathophysiological outcomes. The results obtained will have a high impact on research in this field by shifting the mechanistic focus of GH action from correlation and inferred function to causality. These studies will also serve as a paradigm for the pulsatile hormone action of other endocrine factors that act through complex epigenetic mechanisms.